Frequency domain cross-correlation fluorometry is a known process by which excited light is modulated at a given frequency. The phase shift and the modulation ratio of emission with respect to excitation are measured to obtain the lifetime of the excited state, making use of the cross-correlation technique. This is a versatile tool for determining many subtle characteristics of matter and their interactions. See, for example, the article by E. Gratton and B. Barbieri entitled Multifrequency Phase Fluorometry Using Pulsed Sources: Theory and Applications; Spectostropy, Vol. I, No. 6, pp. 28-38 (1986). See also the article by Gratton, Jameson & Hall entitled Multifrequency Phase and Modulation Fluorometry; Ann. Rev. Biophys. Bioeng. Vol. 13, pp. 105-124 (1984). See also the article by Gratton and Limkeman entitled A Continously Variable Frequency Cross-Correlation Phase Fluorometer With Picosecond Resolution, Biophysical Journal, Vol. 44, pp. 315, 324 (1983). The above articles are given by way of example only, with other articles being available as well.
Fluorometers having continuous frequency domain cross-correlation fluorometric function are commercially available from I.S.S. Inc. of Champaign, Ill., such fluorometers being sold under the trademark GREG. Such a device is capable of measuring and analyzing excitation and emission spectra, fluorescence decays, phase and modulation resolved spectra, time-resolved spectra, and the dynamic depolarization of various materials. As is well known, the apparatus has a highly collimated xenon arc lamp for steady-state measurements and routine lifetime determinations, plus a laser source (UV-visible) for lifetime measurements requiring extremely high sensitivity and accuracy. The modulation of the excitation light is obtained using a wide band electro-optical modulator, specifically a Pockels cell. The Pockels cell is modulated by a direct synthesis frequency synthesizer which provides a signal to the Pockels cell at a first frequency. Another direct synthesis synthesizer provides a signal, in phase coherence with the first synthesizer by driving the two synthesizers from the same quartz crystal. This second synthesizer provides a signal at a second frequency, different from the first frequency, to a pair of detector units set up for detecting luminescence in the sample generated by the modulated light source.
The signal at the output of the detector unit contains a component which is of a frequency that is the difference between the two frequencies, which difference is named the "cross-correlation frequency". The cross-correlation frequency is filtered and processed by a data acquisition unit to provide both the phase shift and the modulation difference of the luminescence of the sample, when compared with the modulated excitation light directed at the sample.
The electronic circuits are designed to filter and process the cross-correlation frequency component only. At the present time, only cross-correlation frequencies of 25 hertz, 31 hertz, and 40 hertz are used. Appropriate phase coherence at these low frequencies are obtained by use of the direct synthesis frequency synthesizers which have a resolution of 1 hertz.
It would be desirable to use phase-locked loop frequency synthesizers because their cost per unit is currently at least two thousand dollars less than each direct synthesis synthesizer unit. However, their use has been, up to the present time, impractical due to the phase noise of the cross-correlation frequencies used in the prior art.
In accordance with this invention, apparatus for frequency domain cross-correlation fluorometry is provided which utilizes phase locked-loop frequency synthesizers. For this reason alone, the cost saving in the apparatus, when compared with the use of direct synthesis synthesizers, may currently be at least four thousand dollars per unit and very probably more than that. Furthermore, phase-locked loop synthesizers operate in a larger frequency range, which increases the lifetime range measurable using cross-correlation frequency domain fluorometers. Moreover, the electronic filter that separates the cross-correlation frequency component of the output signal is cheaper than the corresponding electronics involved with the direct synthesis synthesizers, and is of easier construction. Additionally, the improvements of this invention allow faster measurements, with reduced dead time between two consecutive measurements. This, in turn, greatly facilitates the possibilities in studies of lifetime kinetics measurements.